Project Summary/Abstract The Golgi complex is the central sorting station for nearly a third of all proteins in eukaryotic cells, but how cells regulate the complex flow of material through this organelle remains largely unknown. Protein and membrane traffic at the Golgi is controlled by Arf GTPases that function by recruiting effectors to make outgoing vesicles and tether incoming vesicles. The master regulators that activate Arf GTPase pathways are Arf-GEFs (guanine nucleotide exchange factors). In order to understand the molecular logic of Golgi trafficking, we must understand how the Golgi Arf-GEFs are regulated to make the molecular decision of where and when to activate their substrate Arf proteins. We have uncovered regulatory mechanisms that govern the function of the trans-Golgi network (TGN)-localized Arf-GEF, Sec7. We discovered that Sec7 can switch between autoinhibited and activated states, and is regulated by direct interactions with the activated forms of four different Golgi GTPases: Ypt31/32 (Rab11), Ypt1 (Rab1), Arl1, and Arf1. This collection of interactions represents a previously unappreciated level of crosstalk between these prominent Golgi GTPase pathways. Key questions remain regarding the biochemical basis for Sec7 regulation and the cell biology underpinning these interactions. Furthermore, we have determined that Gea1 and Gea2, the Arf-GEFs that localize to early Golgi compartments, are regulated through mechanisms that are distinct from those of Sec7. Our long-term goal is to determine how cells regulate trafficking at the Golgi complex. In order to both broaden and deepen our mechanistic understanding of the regulation of Arf activation at the Golgi, we propose the following Aims for this project: 1) Investigate the intra-molecular and inter-molecular interactions that regulate Sec7. We will use our established in vitro assays to characterize a newly identified functional link between distinct Sec7 regulatory domains. Using a new assay for measuring Sec7 activity in vivo, we will explore the possibilities that Sec7 integrates multiple GTPase signals and can sense and respond to changes in Golgi cargo load. We will perform an intragenic suppressor screen to identify new functional connections between Sec7 regulatory domains. Finally, we will seek additional structural information regarding the Sec7 C-terminal regulatory domains. 2) Determine the mechanisms regulating localization and activity of the early-Golgi Arf-GEFs Gea1/2. In order to understand how trafficking is regulated at the early- Golgi, we will perform a comprehensive investigation of the mechanisms regulating Gea1/2 membrane localization and activity. We will utilize chimeras and temperature sensitive-mutants to identify and characterize the regions of Gea1/2 regulating localization and activity. We will combine in vitro and in vivo approaches to characterize the roles of Gea1/2 binding partners. Finally, we will seek structural information to explain the basis for Gea1/2 regulation. We expect the outcome of these studies will be new and refined mechanistic models for regulation of trafficking at the Golgi complex.